Targeting of EMILIN-1 and EMILIN-2 to Fibrillin Microfibrils Facilitates their Incorporation into the Extracellular Matrix

Abstract

Elastin microfibril interface-located proteins (EMILINs) 1 and 2 belong to a family of structurally related extracellular glycoproteins with unique functions in the extracellular space, such as modulation of pro-transforming growth factor-β processing, activation of the extrinsic apoptotic pathway, and regulation of Hedgehog and Wnt ligand bioavailability. However, little is known about how EMILINs may exert their extracellular functions. We therefore investigated the spatiotemporal localization and deposition of EMILIN-1 and -2 within the extracellular space. By using immunoelectron and immunofluorescence microscopy together with biochemical extraction, we showed that EMILIN-1 and -2 are targeted to fibrillin microfibrils in the skin. In addition, during skin wound healing and in vitro matrix fiber assembly by primary dermal fibroblasts, EMILIN-1 and -2 are deposited on and coregulated with fibrillin. Analysis of wounds and mouse embryonic fibroblast cultures showed that EMILIN-1 and -2 network formation also requires the presence of fibronectin. Disruption of microfibrils in fibrillin-1-deficient mice leads to fragmentation of the EMILIN-1 and -2 networks, suggesting an involvement of EMILINs in fibrillin-related skin disorders. The addition of EMILINs to the ligand repertoire of fibrillin strengthens the concept of fibrillin microfibrils as extracellular scaffolds integrating cellular force transmission and growth factor bioactivity.

Fibulin-4 deposition requires EMILIN-1 in the extracellular matrix of osteoblasts

“…Similar to our findings with dermal primary fibroblasts 20 , knockdown of one EMILIN in primary calvarial osteoblasts was not affecting fibrillogenesis of the other EMILIN and knockdown of both EMILINs was not affecting fibrillin-1 or fibrillin-2 deposition in primary calvarial osteoblasts or MC3T3-E1 cells (Supplementary Fig. S3, Supplementary Fig.…”

“…A similar dependence on fibronectin for EMILIN-1 assembly was observed in the ECM of E14 mouse embryonic fibroblasts (MEFs) 20 , suggesting that the architecture of the ECM produced by osteoblasts shows similarities to the ECM during embryonic development. However, only simultaneous depletion of both fibrillins in MEFs clearly affected EMILIN-2 matrix deposition 20 .…”

“…We next asked whether EMILIN matrix deposition depends on fibrillin fiber assembly as previously demonstrated for cultured dermal fibroblasts 20 . Similar to our findings with dermal primary fibroblasts 20 , knockdown of one EMILIN in primary calvarial osteoblasts was not affecting fibrillogenesis of the other EMILIN and knockdown of both EMILINs was not affecting fibrillin-1 or fibrillin-2 deposition in primary calvarial osteoblasts or MC3T3-E1 cells (Supplementary Fig.…”

“…Our previous studies in murine dermis revealed that the ECM deposition of EMILIN-1 and -2 crucially depends on the successful assembly of fibrillin microfibrils 20 . Therefore we were curious to investigate whether this interdependence also exists in other tissues.…”

“…We recently demonstrated that both EMILIN-1 and EMILIN-2 are targeted to fibrillin microfibrils in the skin and that fibrillin-1 is required for the proper deposition of EMILINs within the extracellular space 20 . This indicates that EMILIN-1 and -2 alterations may play a role in the pathomechanisms of the fibrillinopathies, where microfibril destabilization due to fibrillin-1 or -2 deficiency leads to multisystemic features characterized by reduced tissue integrity and global activation of growth factor signaling 21 .…”